Sieving media from planar arrays of nanoscale grooves, method of making and method of using the same

ABSTRACT

Disclosed herein are an apparatus and a method for separating molecules on the basis of size and or structure, and to a method of making the apparatus. Generally, the separation method includes passing a fluid comprising particles having different effective molecular diameters through a plurality of open, nanoscale channels disposed in surfaces of substrates. The method also includes obtaining a plurality of fractions of the passed fluid such that each of the fractions includes a major portion containing particles having similar size and shape and substantially free of particles having larger size and shape. The apparatus includes first and second substrates each of which has a surface containing a plurality of open, nanoscale channels disposed therein. The surfaces are bonded together such that each of the channels of the first substrate is in fluid communication with at least two of the channels of the second substrate and is misaligned relative to the channels of the second substrate. Interferometric lithography and anodic bonding or flip-chip bonding techniques can be used to make the apparatus.

RELATED APPLICATIONS

This application is a Divisional of and claims priority to U.S. patentapplication Ser. No. 10/738,465, filed Dec. 17, 2003, entitled “Sievingmedia from planar arrays of nanoscale grooves, method of making andmethod of using the same,” now pending, the disclosure of which isincorporated by reference in the disclosure of this application.

BACKGROUND OF THE DISCLOSURE

1. Field of Invention

The invention relates generally to an apparatus and method forseparating molecules on the basis of size and/or structure, and to amethod of making the apparatus.

2. Brief Description of Related Technology

It is important in the chemical and biological sciences to be able toseparate different molecules from one another. Accurate and preciseseparation is especially important where the molecules are present inonly a small volume solution, such as, for example, in the context ofanalytical and diagnostic testing. There remains a need to improve theefficiencies of such separations and, thereby, the convenience toresearchers working in the chemical and biological sciences.

Generally, molecular separation techniques can include the use of amatrix (or membrane) where molecular transport and filtration occurperpendicular to the surface of the matrix. In such techniques, onlythose molecules having a precise, pre-determined molecular weight and/orstructure pass through the matrix. These separation techniques, however,are limited. For example, biomolecules may not be amenable to separationby such techniques because, for example, they may undesirably reactwith, or be rendered inactive by, the separating matrix. Even wherebiomolecules are amenable to these techniques, the separation can beimprecise, inaccurate, and/or difficult to reproduce due tobatch-to-batch variations in the manufacture of the matrices. Poorseparation efficiency and/or loss of sample volume also can beencountered.

In the biological sciences, gel fractionation or electrophoresis hasbeen found to be a useful technique to separate and identifybiomolecules such as, for example, proteins. Generally, in gelelectrophoresis, the gel consists of a matrix of entangled polymerchains, intermixed with a buffer solution. A large number ofinterconnected pores are present within the matrix. A solution ofproteins having a net electrical charge are placed in the matrix andtravel through the pores under the influence of an electric field.Typically, a charged protein will move towards the pole with a chargeopposite to that carried on the protein. The free-solution mobilities ofdenatured proteins are identical. In the presence of the gel matrix,however, protein mobilities tend to differ because the larger theprotein, the more likely it will encounter a physical restriction in thematrix (either between or within the pores), thus retarding theprotein's progress through the matrix relative to smaller proteins. Thefrictional force of the gel material acts as a protein sieve (or, moregenerally, a molecular sieve) separating the proteins by size. The rateat which a protein migrates through the electric field and gel matrixdepends upon, for example, the strength of the field, size and shape ofthe protein, relative hydrophobicity of the sample in which the proteinis present, and on the ionic strength and temperature of the buffer inwhich the protein is moving. Thus, as smaller proteins should movethrough the matrix faster than large proteins, the proteins becomeseparated with fast moving bands of small proteins at the front and slowmoving bands of larger proteins trailing behind.

One particular type of gel fractionation is two-dimensional (2-D) gelfractionation, which is useful for separating and identifying proteinsin a sample by displacement in two dimensions oriented at right anglesto one another. Two-dimensional gel fractionation is generally used as acomponent of proteomics and is a common step used to isolate proteinsfor further characterization by, for example, mass spectroscopy. Thisfractionation technique permits component proteins of the sample toseparate over a larger area, increasing the resolution of each componentprotein. IEF (isoelectric focusing) and SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) comprise the two dimensions in a 2-Dgel fractionation. In a first dimension, IEF fractionates biomoleculeson the basis of pI values. In the subsequent, second dimension, SDS-PAGEfurther fractionates the previously-fractionated biomolecules based onsize-charge ratios, which roughly correspond to a fractionation based onmolecular weights.

Despite its widespread use, however, 2-D gel fractionation has itslimitations. For example, it is not particularly good at resolvingproteins or peptides having a low molecular mass as these often migratethrough a polyacrylamide gel too rapidly. 2-D gel fractionation also isunsuitable for many proteins, such as hydrophobic proteins, because theproteins often interact with the gel matrix or otherwise undesirablyreact rendering subsequent analysis of the proteins difficult orimpossible. Even when and where the proteins do not undesirably interactor react, it is often difficult to remove them from the gel, thuscompromising the quality of any subsequent analysis of the protein.Another particular limitation is that the fractionation takes a longtime to perform and requires extensive manual handling and attention,which makes it a long and laborious process requiring a skilledtechnician or scientist to master and perform. Performing thefractionation is very much an art, requiring much experimentation tofind the correct conditions for sample preparation, focusing times, etc.Moreover, it is often difficult to reproduce the exact processingconditions under which multiple gels are made and, therefore, there canbe inconsistencies between the various gels. Furthermore, gels canprovide only limited resolution, which is often inadequate for certainmolecular separation and analytical operations, and are often notre-usable. Still further, the gel material can disadvantageouslydegrade—polyacrylamide gel is a neurotoxin having a short shelf-liferequiring that it be prepared just prior to use, and having propertiesthat vary from batch to batch. Additionally, and given the foregoinglimitations, the technique is often inadequate and/or whollyinappropriate for use in an integrated separation and analysis system.Though there have been advances to improve on certain of the foregoinglimitations, many of the limitations still remain.

Alternatives to 2-D gel fractionation include techniques that utilizeartificial gel media. In contrast to polyacrylamide gels where thesieving matrix is defined by random arrangement of long-chain polymers,the sieving matrix in artificial gels is defined by microfabricationand/or nanofabrication. Thus, the dimensions and topology of the sievingmatrix in an artificial gel can be controlled and measured moreprecisely, and can be mass-produced more easily. For example,conventional photoresist-based lithography can be used to etch a patternof obstacles on a silicon substrate (floor), which can be sealed with aglass or elastomeric ceiling layer to form a sieve through which asolution of molecules can be electrophoresed. Similarly, monolithicstructures can be prepared with a sacrificial layer sandwiched between adielectric floor and ceiling layers to define a working gap, wherein thesacrificial layer represents what will be the open space in the finishedstructure and, thus, the negative of the desired pattern of obstacles isetched into it. After the floor, ceiling, and retarding obstacles havebeen put in place, the sacrificial layer is removed by a wet chemicaletch, leaving a working gap whose vertical dimensions are defined by thethickness of the removed layer. Due presumably to critical dimensionlimitations, however, only nucleic acid separations have been reportedwith these structures. Protein albumin is about four nanometers (nm)wide and about fifteen nanometers in length and, therefore, is too smallto interact physically with patterned structures having 100 nm diameterpillars on a 200 nm pitch. Other techniques contemplate the use ofelectrochromatography, in situ casting of sieving media within preformedchannels of a substrate, and the use of porous materials such as poroussilicon as a porous media. Notwithstanding these advances, there remainlimitations not adequately addressed in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the disclosure, reference should bemade to the following detailed description and accompanying drawingswherein:

FIG. 1 is an enlarged, top view of a surface of a substrate having aplurality of nanoscale channels disposed therein;

FIG. 2 is an enlarged, cross-sectional view of the substrate taken alongline 2-2 in FIG. 1;

FIG. 3 is an enlarged, exploded view of a portion of an apparatusshowing its constituent parts;

FIG. 4 is an enlarged, fragmentary plan view of the apparatus with thenanoscale channels disposed in each substrate shown in phantom; and,

FIG. 5 is an enlarged, cut-away view of the apparatus showing the pathof a material traversing the nanoscale channels.

While the disclosed apparatus and methods are susceptible of embodimentsin various forms, there are illustrated in the drawings (and willhereafter be described) specific embodiments of the invention, with theunderstanding that the disclosure is intended to be illustrative, and isnot intended to limit the invention to the specific embodimentsdescribed and illustrated herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein the term “nanoscale channel” refers to any void space ina surface of a substrate having a diameter in at least one direction ofabout one to about 500 nm. When referring to the channel, the term“diameter” is used in its ordinary sense, i.e., the distance across andthrough the middle of the channel, perpendicular to the axis of thechannel, and parallel to the plane of the substrate in which the channelis disposed. When referring to a channel(s), however, the term“diameter” is not intended to limit the cross-sectional shape of thechannel(s) to a circle, as any channel shape can be employed. Thus, theterm “diameter” as used herein also includes “equivalent diameter” asdefined in Table 5-8 of “Perry's Chemical Engineers' Handbook,” at p.5-25 (6^(th) Ed., 1984) (see also 7^(th) Ed., 1997, at pp. 6-12 to6-13). As used herein, the term “array” refers to any arrangement ofnanoscale structures or channels.

Disclosed herein is an apparatus comprising first and second substrates,each of the substrates having a surface containing a plurality of open,nanoscale channels disposed therein. The surfaces are bonded togethersuch that each of the channels of the first substrate is in fluidcommunication with at least two of the channels of the second substrateand is misaligned relative to the channels of the second substrate.

The channels can have equivalent and constant cross-sectional areaswithin a range of about one square nanometer (nm²) to about 10,000 nm²,and more preferably, about 10 nm² to about 1000 nm². Alternatively, thechannels can have equivalent and variable cross-sectional areas within arange of about 1 nm² to about 10,000 nm², and more preferably, about 10nm² to about 1000 nm². Thus, in such an embodiment, a first portion ofthe channel can have a cross-section areas of about 10,000 nm², forexample, while a second portion of the channel can have across-sectional areas of about 1 nm², for example. Benefits of suchvariable cross-sectional areas within the same channel can be realizedwhen resolution of various, different-sized particles is desired. Thechannels within each substrate should be parallel to each other andshould traverse an entire length of the surface in which they aredisposed. Preferably, the channels within each substrate are spacedequidistant from each other, though they need not be. Given thecross-sectional areas of the channels and the intended use of theapparatus, each of the surfaces in which the channels are disposedpreferably contains at least about 1000 channels to about ten millionchannels.

Generally, the substrates can be constructed of any material that isamenable to patterning of nanoscale channels and capable of being bondedtogether. Preferably, however, the first and second substrates can bemade from one or more materials selected from the group consisting ofquartz, silica, silicon, porous silicon, polysilicon, and porouspolysilicon. More preferably, one of the materials of construction isquartz. Suitable silicon materials include porous silicon.

The apparatus preferably also includes third and fourth substratesbonded to edge surfaces of each of the first and second substrates. Theedge surfaces should be substantially perpendicular to the channels.

Preferably, the third and fourth substrates can be made from one or morematerials selected from the group consisting of quartz, silica, silicon,porous silicon, polysilicon, porous polysilicon, and silicon oxynitride.More preferably, one of the materials of construction is siliconoxynitride.

As previously noted, each of the channels of the first substrate ismisaligned relative to the channels of the second substrate. Themisalignment can be defined by an angle, which itself is defined by anintersection of a channel of the first substrate and a channel of thesecond substrate. Preferably, the channels of the first substrate aremisaligned relative to the channels of the second substrate by an angleof about 0.05° to about 45°, more preferably about 0.05° to about 15°,highly preferably, about 0.1° to about 10°, and even more highlypreferably about 0.5° to about 5°.

Generally, the apparatus can be made by patterning an array of open,nanoscale channels on a major planar surface of each of a firstsubstrate and a second substrate. The channeled surfaces can be bondedtogether such that each of the channels of the first substrate is influid communication with at least two of the channels of the secondsubstrate, and such that the each of the channels of the first substrateis misaligned relative to the channels of the second substrate. Apreferred method of bonding includes suitable flip-chip bonding methods.Edge surfaces of each of the bonded first and second substrates can becapped with one or more cap substrates bonded to each edge surface,wherein the edge surfaces are substantially perpendicular to thechannels.

There are numerous suitable methods of patterning an array of open,nanoscale channels on a surface of a substrate. Examples of suchsuitable methods include lithography methods such as, for example,interferometric lithography (“IL”), immersion interferometriclithography, electron beam lithography, scanning probe lithography,nanoimprint, extreme ultraviolet lithography, and X-ray lithography.Generally, IL is a preferred method of patterning the nanoscalechannels.

Generally, lithography is a highly-specialized printing process used tocreate detailed patterns on a substrate, such as a silicon wafer. Animage containing a desired pattern is projected onto the wafer, which iscoated by a thin layer of photosensitive material called “resist”. Thebright parts of the image pattern cause chemical reactions which, inturn, render the resist material soluble, and, thus, dissolve away in adeveloper liquid, whereas the dark portions of the image remaininsoluble. After development, the resist forms a stenciled patternacross the wafer surface, which accurately matches the desired pattern.Finally, the pattern is permanently transferred into the wafer surface,for example by a chemical etchant, which etches those parts of thesurface unprotected by the resist.

Interferometric lithography (“IL”) generally refers to a process oflithography where two or more mutually coherent light waves (or beams)interfere to produce a standing wave, which can be recorded in aphotoresist. More specifically, in IL, a sinusoidal standing wavepattern of light intensity is produced by interference at the region ofintersection of two coherent light beams. A photoresist-coated substratepositioned at the point of intersection undergoes exposure, printing aperiodic, line-space pattern whose period (P) is determined by thewavelength of light (λ) and the angle of intersection (A) of the lightbeams (i.e., P=λ/2 sin(A)). The angle (A) should be sufficiently largeto produce an interference pattern that has a high spatial frequency.The resulting interference pattern should have nanoscale dimensions.

Examples of suitable IL techniques that can be used to pattern the arrayof channels are described in, for example, Brueck et al. U.S. Pat. No.5,705,321. Generally, the photoresist-coated substrate is prepared bydepositing a thin, etch-mask layer on a silicon substrate (wafer); thendepositing a thin, photoresist layer on top of the etch-mask layer.Thereafter, the photoresist layer is exposed to the periodic pattern oflines using fine-line IL optimized to yield the appropriate nanoscaledimension of unexposed photoresist. The photoresist is developed toremove the exposed photoresist. The photoresist pattern is nexttransferred into the etch-mask using an etching process (over-etchingthe etch-mask at this point can undesirably undercut the etch mask andfurther narrow the etch mask pattern). The remaining photoresist is thenremoved. A highly anisotropic etching process, such as with potassiumhydroxide, for example, can be used to etch the exposed substrate (e.g.,a silicon (Si) substrate), in which case the lines of the periodicpattern should be aligned with the {111} Si directions prior tophotoresist exposure. In this process, the {111} Si surfaces are almosttotally unetched and, thereby leave very narrow, quantum-sized Si wallswith a very high aspect ratio. If reactive-ion or ion-milling etchprocesses are used instead of potassium hydroxide, then it is notnecessary to pre-align the pattern with the {111} Si directions. Theremaining etch mask then can be removed, leaving an all Si surface,which can be oxcized. The same basic method can be used to fabricatemore complex structures by the use of multiple-exposure IL and/orcombining IL with conventional optical lithography either during thephotoresist exposure step or within multiple iterations of portions ofthe process.

The complex interference pattern produced on the photoresist layer orlayers can be varied by rotating and/or translating the substrate,changing the angle (A), varying the number of exposures and/or theoptical intensity, using a phase-amplitude mask in one or bothilluminating beams of coherent radiation, and any combination of theforegoing. Further flexibility can be attained by a combination of anyof the foregoing variations along with suitable optical imaginglithography techniques.

Though there are many suitable methods of patterning the nanoscalechannels, IL represents one of the more convenient and preferred methodsof patterning nanostructured features because it can be used to generatethe entire pattern in one, parallel step and is not a serial writingtechnique. Other parallel techniques (e.g., imprint lithography) relyupon a primary patterning technique to generate a master thatsubsequently can be used to produce replicas of nanostructured featuresin a parallel fashion. The use of IL to pattern an array of nanoscalechannels has additional advantages over other techniques (such astraditional acrylamide gel polymerization) since it is capable ofcreating highly-ordered structures, provides the possibility of creatingmacroscopic arrays of continually varying size or chemistry across onedimension, is highly reproducible, can be carried out rapidly overlarger macroscopic areas at low cost (low relative to electron-beamlithography, for example), and can be more easily implemented in thecreation of complex, integrated separation systems that are disposableor reusable. Furthermore, the use of lithographically-defined-separationmatrices lends itself to simple implementation of these matrices intomulti-level, 3-dimensional separation devices in which differentscreening mechanisms allow enhanced separations. Additionally, IL can beused to easily generate arrays of nanostructures (protrusions orchannels) whose dimensions vary semi-continuously in the plane ofsurface of the material being patterned. Once the surface of thesubstrate has been patterned with the desired pattern of nanoscalechannels, the patterned surfaces is bonded with another similarlypatterned surface. Thus, the formed apparatus aims to eliminate some ofthe current limitations by the fabrication of highly-uniform andaccurately-reproducible nanoscale separation systems prepared by nano-and microlithography.

A purpose in bonding the substrates together is to create intimate,physical contact between the surfaces of the substrates such that thesolution and any particles therein that are traversing the channelsremain confined to the void space defined by the channels. Suitablemethods of bonding the patterned (or channeled) surfaces togetherinclude, but are not limited to, anodic bonding methods and flip-chipbonding methods capable of mating the surfaces such that each of thechannels of the first substrate is in fluid communication with at leasttwo of the channels of the second substrate, and such that the each ofthe channels of the first substrate is misaligned relative to thechannels of the second substrate. Both anodic bonding and flip-chipbonding methods are known by those skilled in the art. Generally,flip-chip bonding (also known in the art as direct chip attach (DCA)) isa direct electrical connection of face-down (“flipped”) electroniccomponents onto substrates, circuit boards, or carriers, by mean ofconductive bumps on chip bond-pads. In contrast, wire bonding usesface-up chips with a wire connection to each chip bond-pad.

Flip-chip bonding typically comprises bumping a first substrate (orwafer), attaching the bumped substrate to a second substrate, andfilling any remaining void space between surfaces of the substrate witha filler material, such as an electrically-non-conductive material,keeping in mind that the filler material should not fill the channels ofthe substrate. In the electronics arts, the bump can serve manypurposes, such as providing a path for transferring an electric chargeor heat from one substrate to another. Here, however, the bumpadvantageously provides a mechanical mounting to assist in attaching onesubstrate to another. The bump can be prepared by a variety of methodsincluding, for example, those using solder or stud-bumping techniques,vacuum deposition, electroplating, and adhesives. Flip-chip bonding isadvantageous because if offers a high-speed, low-cost assembly methodand results in a suitably rugged bond. Given the flip-chip bondingprocessing conditions, and the intended use of the formed apparatus, oneskilled in the art can appropriately select the materials ofconstruction for use in bonding the various substrates together.

As noted herein, cap substrates can be bonded to edge surfaces of thealready-bonded first and second substrates to prevent the samplesolution from entering or exiting the exposed edges of the apparatus ina direction parallel to the nanoscale channels, and to constrain thesolution into and out of the apparatus in a direction roughlyperpendicular to the direction of the channels. Generally, the capsurface is constructed of silicon oxynitride. The cap surface can bebonded with the aid of an adhesive suitable for attaching a siliconoxynitride surface, for example, to the edge surfaces of thealready-bonded first and second substrates.

Once the cap substrates have been appropriately bonded to the edgesurfaces, the apparatus can be suitably attached to or otherwiseincorporated into a device capable of introducing an electric field anda solution of molecules.

The end result of these manufacturing steps is an apparatus whosenanoscale channels are freely accessible to solution added along theuncapped edges of the 2-chip stack. Hence, conventional electrophoresisand other forms of chromatography can be performed within thenanochannels. The apparatus can be used by filling the interior space(e.g., via capillary action) with a solution containing the molecules tobe separated, and then applying an electric field along a directionroughly perpendicular to the channels. As previously noted, a chargedmolecule will migrate with (or against) the general direction of thisapplied field. The actual path of any given charged molecule will bequite torturous, as explained hereinafter.

Referring now to the drawing figures, wherein like reference numbersrefer to the identical or similar elements in the various figures, FIG.1 is an enlarged, top view of a surface of a substrate having aplurality of nanoscale channels disposed therein. More specifically,FIG. 1 shows a substrate 10 having a surface 12 on or within which aredisposed nanoscale channels 14. Edge surfaces (not shown) of thesubstrate 10 are capped with cap substrates 16 and 18. As shown in FIG.1, each of the channels 14 has a constant cross-sectional diameter andeach appears to be spaced equidistant from one another. as previouslynoted, the channels need not have a constant cross-sectional diameter orbe spaced equidistant from one another. FIG. 2 is an enlarged,cross-sectional view of the substrate 10 taken along line 2-2 in FIG. 1.

FIG. 3 is an enlarged, exploded view of a portion of an apparatus 30showing its constituent parts. As shown, the apparatus 30 includes thesubstrate 10 and a second substrate 20 having a surface 22 on or withinwhich are disposed nanoscale channels 24. Edge surfaces 26 and 28 of thesubstrates 10 and 20, respectively, are capped with cap substrates 16and 18. When the substrates 10 and 20 are mated, the apparatus 30 isformed as edges X-X′ and Z-Z′ meet and cap substrates 16 and 18 arebonded to the edge surfaces 26 and 28.

FIG. 4 is an enlarged, fragmentary plan view of a portion of the formedapparatus 30 with the nanoscale channels 14 and 24 disposed in eachsubstrate shown in phantom. As shown, each of the channels 14 of thefirst substrate 10 is misaligned relative to each channel 24 of thesecond substrate 20. The misalignment is defined in FIG. 4 by an angle(α), which itself is defined by the intersection of a channel 14 of thefirst substrate 10 and a channel 24 of the second substrate 20.

FIG. 5 is an enlarged, cut-away view of the apparatus showing the pathof a material traversing the nanoscale channels 14 and 24, as depictedby the arrows. More specifically, shown in FIG. 5 is the apparatus 30comprising the mated substrates 10 and 20, and the nanoscale channels 14and 24 disposed therein. With the application of a force, such as apressure or an electric field, molecules within a solution (depicted bythe arrows in FIG. 5) can traverse the tortuous path created by thenanoscale channels 14 and 24. Moreover, the molecules are constantlyzig-zagging through an interior space comprised almost entirely ofwedge-shaped cracks. These cracks are the molecular-scale physicalconstrictions that impart a sieving capability to the 2-chip stack. Thespeed at which a particular molecule traverses from one end of theapparatus 30 to the other will, of course, depend upon the molecularweight and structure, as described above.

The formed apparatus is useful to resolve the various molecules presentin a solution. Higher resolution can be obtained where the apparatus isused in combination with any one or more of the following mechanisms:affinity interaction (molecular recognition), asymmetric diffusion,electrophoretic mobility, entropic trapping, hydrophobic interaction,isoelectric point, and size exclusion.

The disclosed apparatus is useful to separate particles within a fluidhaving different effective molecular diameters into discrete portionscharacterized by common effective molecular diameter. Such particles areseparated on the basis of the ability of particles having a smallereffective molecular diameter to pass through the apparatus channels morequickly than those having larger effective molecular diameters. Whereparticles have substantially equivalent molecular diameters, thosemolecules that are shorter in length should pass through the apparatusmore quickly than those molecules that are longer in length. Higherresolution can be obtained where the apparatus is used in combinationwith any one or more of the following mechanisms: affinity interaction(molecular recognition), asymmetric diffusion, electrophoretic mobility,entropic trapping, hydrophobic interaction, isoelectric point, and sizeexclusion.

Suitable fluids that can pass through the apparatus include biologicallyderived materials such as, for example, peptides, polypeptides,proteins, antigens, antibodies, nucleotides, oligonucleotides,polynucleotides, aptamers, DNA, RNA, carbohydrates, complexes thereof,and suitable buffers. Fluids also can include non-biologically-derivedmaterials such as, for example, synthetic polymers.

A buffer is a defined solution that resists change in pH when a smallamount of an acid of base is added or when the solution is diluted. Forexample, the pH of the blood in a healthy individual remains remarkablyconstant at 7.35 to 7.45 because the blood contains a number of buffersthat protect against pH change due to the presence of acidic or basicmetabolities. From a physiological viewpoint, a change of +0.3 or −0.3pH unit can be considered to be extreme. Many biological reactions ofinterest occur in the pH range of 6 to 8. Specific enzyme reactions thatmight be used for analyses may occur in the pH range of 4 to 19 or evengreater. Thus, buffers are very useful for maintaining the pH at anoptimum value. The proper selection of buffers for the study ofbiological reactions or for use in clinical analyses can be critical indetermining whether of not they influence the reaction.

Proteins are amphoteric compounds; their net charge therefore isdetermined by the pH of the medium in which they are suspended. In asolution having a pH above the protein's isoelectric point, a proteinhas a net negative charge and migrates towards the anode in anelectrical field. Below its isoelectric point, the protein is positivelycharged and migrates towards the cathode. The net charge carried by aprotein is independent of its size, meaning that the charge carried perunit mass (or length, given proteins and nucleic acids are linearmacromolecules) of molecule differs from protein to protein. Thus, at agiven pH and under non-denaturing conditions, the electrophoreticseparation of proteins is determined by both size and charge of themolecules. In contrast to proteins, nucleic acids remain negative at anypH used for electrophoresis and carry a fixed negative charge per unitlength of molecule, provided by the phosphate group of each nucleotideof the nucleic acid. Thus, electrophoretic separation of nucleic acidsproceeds strictly according to size.

Sodium dodecyl sulphate (SDS) is an anionic detergent that is used todenature proteins by “wrapping around” the polypeptide backbone ofproteins—and SDS binds to proteins fairly specifically in a mass ratioof about 1.4:1. In so doing, SDS confers a negative charge to thepolypeptide in proportion to its length (the denatured polypeptidesbecome “rods” of negative charge cloud with equal charge or chargedensities per unit length). It is usually necessary to reduce disulphidebridges in proteins before they adopt the random-coil configurationnecessary for separation by size, such as, for example, with2-mercaptoethanol or dithiothreitol. In denaturing SDS-basedseparations, therefore, migration is determined not by intrinsicelectrical charge of the polypeptide, but by molecular weight.

Thus, if a mixture of SDS-complexed proteins in a suitable buffer iselectrophoresed through the 2-chip stack, the larger the protein, themore likely it will encounter a restriction, and hence be retardedrelative to a smaller protein. Proteins will elute from the chip in theorder of size, the smallest first, and the largest last. The separatedproteins can be further analyzed as desired.

The foregoing description is given for clearness of understanding only,and no unnecessary limitations should be understood therefrom, asmodifications within the scope of the disclosure may be apparent tothose having ordinary skill in the art.

1. A method comprising: (a) passing a fluid comprising differentcomponents having different effective molecular diameters from end toend of an apparatus having at least about 1000 to about ten millionopen, nanoscale channels disposed in surfaces of a first and secondsubstrates, the first and second substrates bonded together such thateach of the channels of the first substrate is in fluid communicationwith at least two of the channels of the second substrate and ismisaligned relative to the channels of the second substrate, and each ofthe channels of the second substrate is in fluid communication with atleast two of the channels of the first substrate, and wherein the fluidcommunication between channels creates a continuous nonlinear pathway inwhich the fluid passes alternatingly between the channels of the firstsubstrate and the channels of the second substrate; (b) obtainingdifferent fractions of the passed fluid, each of the fractionscomprising a major portion comprising components having similar size andshape and substantially free of components having a different size andshape wherein the size and shape of the major portion of components ofeach of the fractions is different from the size and shape of the majorportions of the components of the other fractions.
 2. The method ofclaim 1, wherein the channels have equivalent and constantcross-sectional areas within a range of about 1 square nanometers (nm²)to about 10,000 nm².
 3. The method of claim 1, wherein the channels haveequivalent and variable cross-sectional areas within a range of about 1nm² to about 10,000 nm².
 4. The method of claim 1, wherein each of thechannels traverses an entire length of the surface.
 5. The method ofclaim 1, wherein the channels of the first substrate are parallel toeach other, and the channels of the second substrate are parallel toeach other.
 6. The method of claim 1, wherein the channels of the firstsubstrate are spaced equidistant from each other, and the channels ofthe second substrate are spaced equidistant from each other.
 7. Themethod of claim 1, wherein the first and second substrates comprise oneor more materials selected from the group consisting of quartz, silica,silicon, porous silicon, polysilicon, and porous polysilicon.
 8. Themethod of claim 7, wherein the first and second substrates comprisequartz.
 9. The method of claim 5, further comprising third and fourthsubstrates bonded to edge surfaces of each of the first and secondsubstrates, the edge surfaces being substantially perpendicular to thechannels.
 10. The method of claim 9, wherein the third and fourthsubstrates comprise one or more materials selected from the groupconsisting of quartz, silica, silicon, porous silicon, polysilicon,porous polysilicon, and silicon oxynitride.
 11. The method of claim 10,wherein the third and fourth substrates comprise silicon oxynitride. 12.The method of claim 1, wherein the channels of the first substrate aremisaligned relative to the channels of the second substrate by an angleof about 0.05° to about 45° , the angle defined by an intersection of achannel of the first substrate and a channel of the second substrate.13. The method of claim 1, wherein one or more of the substratesadditionally includes electrodes capable of creating an electric fieldalong at least a portion of the nonlinear path traveled by a liquidpassing through the continuous nonlinear pathway.